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					Original Article                                                                                                   59

                       Wandee Gritsanapan1,∗ and Peeranuch Mangmeesri2
     1Department   of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand
         2Faculty   of Medicine Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand

           ABSTRACT: Senna alata (L.) Roxb. is a medicinal plant of which the leaves have long been
           used as a laxative and antifungal drugs. It is one of the plants recommended to be used in
           primary health care in Thailand and is included in the Thai traditional household drug list for
           laxative and antifungal herbal drugs. It is also listed in the Lists of National Herbal Drugs of
           Thailand. The leaves contain anthraquinones both aglycone and glycoside forms. The content
           of anthraquinones in the leaves are varied mainly according to cultivating locations and
           harvesting period. It is necessary to control the quantity of antraquinones in the leaves and leaf
           extracts of this plant for good quality of laxative and antifungal raw materials. This work was
           conducted to standardize 80% ethanolic extracts prepared by the appropriate maceration
           method of S. alata leaves collected from 10 different locations in 4 major parts i.e. the North,
           North-East, Central and South of Thailand. The extract ratio was 3-4:1, while the
           characteristics of the extracts were dark brown semi-solid with characteristic odour. The
           extract gave a positive Bontrager’s test and its TLC-fingerprints showed 2 major anthraquinone
           components as rhein and aloe-emodin. Total anthraquinones content in the extract analyzed
           by UV-vis spectrophotometric method was not less than 2%w/w (average 2.48% w/w)
           calculated as rhein. Loss on drying of the extract should not more than 6%w/w while the
           extract was slightly soluble in water but freely soluble in 95% ethanol. For heavy metals
           contamination, the extracts should not contain arsenic, while lead, mercury and cadmium
           should not be found more than 2, 0.1 and 0.2 ppm, respectively. Total aerobic bacteria count
           and total yeast and mold count should not exceed 100 and 1000 cfu/g, respectively while none
           of pathogenic bacteria was found in the extract. Standardized S. alata leaf extracts could be
           used as a good quality raw material containing anthraquinones for laxative and antifungal
           pharmaceutical preparations.
           Keywords: Senna alata, Cassia alata, Fabaceae, standardization, rhein, anthraquinone

INTRODUCTION: Senna alata (L.) Roxb. or                      total anthraquinone glycosides content in the
Cassia alata L. is a medicinal plant in the family           leaves of nine Cassiae i.e. C. siamea, C. fistula, C.
Fabaceae which has been known in Thai                        alata, C. surattensis subsp. surattensis, C. grandis,
language as Chumhetthet. The English names are               C. spectabilis, C. bakeriana, C. sophera and C. tora
Candelabra bush and Ringworm bush1). It is a                 collected in Summer, Winter and Rainy seasons
native plant of South America and can be found               from the Central areas of Thailand and found that
widely in tropical region, up to 1500 m, on waste            the leaves of C. alata collected in Winter and
places often along ditches2). In Indonesia,                  Summer contain the highest amount (1.24% dry
Philippines and Thailand, this plant can be found            weight) of total anthraquinone glycosides. We also
all over the countries, sometimes cultivated for             found that most of Cassia leaf samples containing
medicinal purposes3). After 3 months of planting,            the maximum content of anthraquinone
leaves are ready for harvest, but the best period            glycosides are the samples collected in Summer
for the best quality is about 6-7 months after               (March-June) and Winter (November-February)
planting4). Fresh or dried leaflet of S. alata has           seasons. S. alata is one of the plants recom-
been used as folk medicines in many countries for            mended to be used in primary health care in
treatment     of constipation, stomach pain,                 Thailand and has been listed in Thai traditional
ringworm and skin diseases5, 6). The leaf contains           household drug list for laxative and antifungal
anthraquinones both aglycone and glycoside                   drugs. At present, S. alata is included in the List
forms i.e. rhein, aloe-emodin, chrysophanol,                 of Herbal Medicinal Products A.D. 2006 of
glycosides of rhein, emodin, physcione and                   Thailand12). According to the Standard of ASEAN
sennosides A, B, C, D7-10) while rhein is a major            Herbal Medicine4) and Thai Herbal Pharmacopoeia
component11). Gritsanapan et al11) investigated              (THP)13), S. alata leaves should contain not less

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                                                                                          J Health Res 2009, 23(2):59-64
60                                                                                         Original Article

than 0.5 and 1.0 % dry weight of total                  Marceration: The powdered leaves of S. alata
hydroxyanthracene derivatives calculated as             (10.0 g ) were macerated with 80% ethanol (100
rhein-8-glucoside, respectively. The normal way of      ml). The extraction was repeated until exhausted
using S. alata for laxative is that 12 fresh or dried   (tested by Borntrager’s reaction) and the
                                                        maceration extracts were combined, filtered and
leaflets are coarsely cut, boil with 2 glasses of
                                                        evaporated to dryness on a boiling water bath to
water until 1 glass of decoction is obtained and
                                                        yield a maceration crude extract (3.09 g).
strain into a glass. Take the whole decoction as a
single dose when needed. Another way, macerate          Percolation: The powdered leaves of S. alata
1-2 teabags of 3 g of dried powdered leaves in a        (10.0 g ) were moistened with 80% ethanol (30 ml)
cup of boiling water for 2-5 minutes, and take the      for 15 minutes. The moistened material was put
infusion at bed time14,15).                             in a percolator and 80% ethanol was added. The
    Extraction method and extracting solvent are        percolation was adjusted at a rate of 1-3 ml/min
important for quantity and quality of the extracts.     until the extraction was exhausted. The extracts
Appropiate extraction method for each plant             were combined, filtered and evaporated to dryness
should be investigated in order to promote the          on a boiling water bath to yield a percolation
highest amount of active components. Thus, this         crude extract (2.26 g).
study was conducted to find out the appropriate
                                                        Soxhlet extraction: The powdered leaves of S.
extraction method for S. alata leaves to promote
                                                        alata (10.0 g ) were extracted with 300 ml of 80%
the 80% ethanolic extract containing the
                                                        ethanol in a soxhlet apparatus. The extraction
maximum amount of total anthraquinones and to
                                                        was continued until the extraction was exhausted.
standardize the extracts of S. alata leaves
                                                        Each extract was then combined, filtered and
collected from 10 different locations in the North,
                                                        evaporated to dryness on a hot water bath to yield
North-East, Central and South of Thailand.
                                                        a soxhlet crude extract (2.56 g).
MATERIALS AND METHODS:                                      The extraction method which promoted the
Plant Materials                                         extract containing the maximum content of total
    The leaves of S. alata were collected from 10       anthraquinones would be choosen as the
different provinces in the North, North-East,           appropriate method for further extracting the leaf
Central and South of Thailand (Table 1) during          samples of S. alata collected from various
the Winter (November-February) 2001-2002. The           locations.
plant specimens were identified by comparison           Identification of Anthraquinones
with the herbariums at the Forest Herbarium,                  Borntrager’s reaction was used to detect
Department of National Park, Wildlife and Plant         anthraquinone aglycones in the extract.
Conservation, Ministry of Natural Resources and         Hydrochloric acid (2M) was added to the sample
Environment, Bangkok. The voucher specimens             and the mixture was heated on a hot water bath
(WSA010201-WSA010210) were deposited at                 for 15 minutes, then cooled and filtered. The
Department of Pharmacognosy, Faculty of                 filtrate was extracted with chloroform. The
Pharmacy, Mahidol University, Bangkok, Thai-            chloroform layer was separated and shaken with
land. Each sample was air dried and then dried in
                                                        10% potassium hydroxide solution. The upper
a hot air oven at 50o C for 5 hours. The dried
                                                        aqueous layer becomes pink-red16).
leaves were ground with an electronic mill and
passed through a sieve (20 mesh).                       Quantitative Determination of Total Anthra-
                                                        quinones by a UV-visible Spectrophotometric
Determination of Appropiate Extraction                  Method in S. alata Leaf Extracts
Method for S. alata Leaves
                                                           The extract (0.10 g) was accurately weighed
   Due to the leaves of S. alata contain both
                                                        and dissolved in 30 ml of distilled water. The
anthraquinone aglycones which are soluble in
                                                        analysis procedure was followed the scheme14).
ethanol, and glycosides which are soluble in
                                                        The content of total anthraquinones was
water, so, 80%v/v was used as a solvent for
                                                        determined by a validated UV-vis. Spectrophoto-
extracting S. alata leaf samples.

J Health Res 2009, 23(2):59-64
Original Article                                                                                              61

                          Scheme 1 Quantitative analysis of total anthraquinones4)

metric method17). The UV absorbance was                    80% ethanolic extract. The yield of crude extract
measured at 515 nm.                                        was recorded and the extract ratio (weight of
   The content of total anthraquinones in the              powdered drug : 1 g extract) was calculated.
extract from each extraction method was
                                                           TLC fingerprints
calculated using the linear regression equation of
                                                              A 5.0 μL volume of each sample solution (20
a reference standard rhein. The contents were
                                                           mg/mL) was applied in form of band of width 8.0
expressed as mean ± standard deviation (SD)
                                                           mm on precoated silica gel aluminum plate 60
                                                           F254 (20x10 cm, E-Merck, Germany) using a
   Preparation of 95% Ethanol Extracts of S.               Camag Limonat densitometer. The mobile phase
alata Leaves Collected from Ten Different                  consisting of ethyl acetate: methanol: water 100:
Provinces                                                  17:13 was used. Linear ascending development
   Each sample of S. alata powdered leaves was             was carried out in 20x10 cm twin through glass
exhaustively extracted with 80% ethanol by the             chamber (Camag, Muttenz, Switzerland) saturated
appropriate extraction method (maceration). The            with the mobile phase. The length of running was
combined extract was evaporated to yield a crude           8 cm. The development was carried out twice with

                                                                                     J Health Res 2009, 23(2):59-64
62                                                                                           Original Article

the same mobile phase to get good resolution of          aeruginosa, Salmonella spp. and Clostridium spp.
rhein. The TLC plate was dried with an air dryer.        were also determined.
The anthraquinones were detected by spraying
                                                         RESULTS AND DISCUSSION:
with 10% ethanolic KOH at room temperature
(28o C). The positive result is a pink to red color of   Determination of appropriate extraction method
bands of anthraquinones detected in day light.               Ethanol (80% v/v) was used as an extracting
                                                         solvent for the leaf samples of S. alata due to both
Determination of Total Anthraquinone Content
                                                         anthraquinone aglycones and glycosides can be
in 80% Ethanolic Extracts of S.alata Leaves
                                                         soluble in this solvent, and ethanol is safe and
from 10 Different Provinces
                                                         not expensive. The extraction method which
   The 80% ethanolic extracts of S. alata leaves
                                                         promoted the extract with maximum content of
from different locations were separately analyzed
                                                         total anthraquinones (2.48 ± 0.20% w/w) was
for total anthraquinone content, calculated as
                                                         found to be the maceration while the extracts
rhein, by UV-vis spectrophotometric method as
                                                         from the percolation and soxhlet extraction
described before. The analysis of each sample was
                                                         contained total anthraquinones 2.46 ± 0.31 and
done in triplicate and the content of total
                                                         2.13 ± 0.29%w/w, respectively. When compared
anthraquinone in each sample was reported as
                                                         various extraction methods concerning yields of
mean ± SD.
                                                         crude extracts and total anthraquinone content in
Physical and Chemical Properties of S. alata             the extracts, maceration was found to be the
Leaf Extracts                                            appropriate extraction method. Thus, maceration
Loss on drying determination                             was used for extracting S. alata leaves collected
    Loss on drying of each extract was determined        from 10 deferent locations.
according to the procedure described in Standard         Standardization of 80% ethanolic extracts of
of ASEAN Herbal Medicine (1993). Each sample             S. alata leaves
was done in triplicate and the average of weight             All 80% ethanolic extracts of S. alata leaves
lost on drying was reported.                             were dark brown semi-solid with characteristic
Solubility of extracts                                   odour. TLC fingerprints of all extracts showed the
   The solubility of each extract in 95% ethanol         similar pattern while rhein and aloe-emodin were
and distilled water was investigated at room             found to be major constituents with hRf values
temperature. The level of solubility was recorded        39-44 and 74-79, respectively. The average
according to USP XXVI criteria18).                       extract ratio (crude drug: 1g crude extract) was
Heavy metal residues                                     3.32:1 (3-4:1). The yields of crude extracts from
   Residues of heavy metals (Cd, Pb, As and Hg)          the dried leaves were varied from 27.90 to 31.13%
in the extracts were determined according to the         w/w (Table 1). The contents of total anthra-
American Organization of Analytical Chemists             quinones (total aglycones + total glycosides)
(AOAC) official method of analysis19).                   calculated as rhein in all extracts were not much
                                                         different and found in the range of 2.41 to 2.48
Pesticide residues
                                                         %w/w (average 2.45 % w/w). Compared to
   Pesticides (organochlorines, organophosphates
                                                         standardized senna leaf dry extract which
and pyrethroids) residues in the extracts were
                                                         contains not less than 5.5% and not more than
determined according to AOAC (2006).
                                                         8.0% of hydroxy anthracene glycosides calculated
Microorganism contamination                              as sennoside B20), Senna alata leaf extract
   Total aerobic bacterial count and total fungi         contained about 1/2 to 1/3 time of anthra-
count were determined according to the method            quinones in the standardized senna extract. Loss
described in Standard of ASEAN Herbal Medicine           on drying of the extracts was not more than 6%
(1993). Some pathogenic bacteria i.e. Staphylo-          w/w (average 5.02 % w/w) (Table 2). All extracts
coccus aureus, Escherichia coli, Pseudomonas             were freely soluble in 95% ethanol and slightly

J Health Res 2009, 23(2):59-64
Original Article                                                                                                              63

      Table 1 Yield, extract ratio, and content of total anthraquinones in the ethanolic extracts and dried powder of
      S. alata leaves collected from various locations
                                                       Yield                       Total antraquinones (%w/w)*
          Sample           Location/Part                        Extract ratio
                                                     (%w/w)*                      In extract      In dried powder
             1               Bangkok/C              27.90±0.15     3.58:1         2.41±0.02          0.67±0.08
             2                Phichit/C             29.85±0.08     3.35:1         2.47±0.03          0.74±0.02
             3              Nongkhai/NE             30.95±0.10     3.23:1         2.48±0.30          0.77±0.04
             4             Udonthani/NE             31.20±0.23     3.20:1         2.44±0.10          0.76±0.03
             5         Maha sarakham/NE             30.94±0.12     3.23:1         2.43±0.05          0.75±0.12
             6              Sukhothai/N             30.30±0.25     3.30:1         2.44±0.10          0.74±0.05
             7             Phatthalung/S            29.82±0.17     3.35:1         2.47±0.08          0.74±0.10
             8       Nakhon Si-thammarat/S          29.27±0.31     3.42:1         2.48±0.18          0.73±0.12
             9        Ron Phibun-Nakhon Si-         31.13±0.29     3.21:1         2.47±0.19          0.77±0.17
            10             Surat-Thani/S            29.73±0.17     3.36:1         2.47±0.20          0.73±0.15
          average                                   30.11±0.18     3.32:1         2.45±0.12          0.74±0.09
           *expressed as mean ±SD (n=3)
           C = Central, NE =North-East, N = North, S = South

      Table 2 Characteristics, loss on drying, heavy metal and pesticide residues, and microbial contamination of ethanolic
      extracts of S. alata leaves from 10 locations.
                                                                    Limit allowed for dried      Limit allowed for food
                       Test                          Result
                                                                   herbal raw materials (1)                (2)
       Color                                      dark brown
       Odour                                    characteristics
       Solubility in water                      slightly soluble
       Solubility in 95% Ethanol                 freely soluble
       Average loss on drying (%w/w)               5.02±0.34
       Test with Borntrager’s                       positive
       Heavy metal residues (ppm)
            Cd                                       < 0.17                   < 0.3                         -
            Pb                                       < 2.00                   < 10                          1
            As                                         ND                      <4                           2
            Hg                                       < 0.07                     -                          0.5
       Pesticide residues (ppm)                        ND                    0.01-1                         -
       Microbial contamination (cfu/g)
            Total aerobic bacteria count             < 100                < 5.0 x 105/g

            Yeast and mold count                     < 1000               < 5.0 x 103/g
            Enterobacteria                            non                 < 5.0 x 103/g
            E.coli                                    non                    < 5.0/g
            P. auruginosa                             non                      non
            S. aureus                                 non                    non/g
            Salmonella spp.                           non                   non/10g
            Clostridium spp.                          non                   non/10g
      (1) =           Thai Herbal Pharmacopia Vol. I, 1995
      (2) =            Thai Food Acts, 1986
      ND =             not detectable

soluble in water. Due to no specific limit for                     S. aureus, E. coli, P. aeruginosa, Salmonella spp.
herbal extracts on heavy metal and pesticide                       and Clostridium spp. were found in all the
residues and microbal contamination,the limits                     extracts.
allowed for herbal raw materials and foods were                    CONCLUSION: Maceration was found to be
used as the guidances. The residues of heavy                       appropiate for extraction S. alata leaves. 80%
metals (Cd, Pb, As and Hg) in all extracts of S.                   Ethanolic extracts of the leaves collected from 10
alata leaves were below the allowed limits                         different locations in the North, North-East,
according to THP (Table 2). For pesticide residues,                Central and South of Thailand contained total
organochlorines, organophosphates and          pyre-               anthra-quinones, calculated as rhein determined
throids could not be detected in all extracts (Table               by UV-vis spectrophotometic method, not less
2). Microbial contamination in all extracts was                    than 2.0% w/w (average 2.45±0.12% w/w). TLC
found below the allowed limits according to THP                    fingerprints of all extracts showed the same
(Table 2). Total aerobic bacterial and total fungi                 pattern which rhein and aloe-emodin were main
counts were less than 100 and 1,000 cfu/g,                         constituents. Loss on drying of the extracts was
respectively while no pathogenic bacteria such as                  not more than 6% w/w while the heavy metal

                                                                                                   J Health Res 2009, 23(2):59-64
64                                                                                        Original Article

residues in the extracts were less than the limits     6. Bhat RB, Etejere EO, Oladipo VT. 1990.
                                                       Ethnobotanical studies from the Central Nigeria.
allowed by ASEAN Herbal Medicine and THP.              Econ Bot 44(3): 382-390.
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                                                       Asian J Pharm 3 (1): 10-12.
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                                                       keaw S, 2005. Investigation of alternative
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ACKNOWLEDGEMENT: This research was                     Herbal Medicinal Products A.D. 2006. Bangkok.:
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Thailand.                                              Public Health. 1995. Thai Herbal Pharmacopoeia.
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